# Characterization of the Binding and Inhibition Mechanisms of a Novel Neutralizing Monoclonal Antibody Targeting the Stem Helix Region in the S2 Subunit of the Spike Protein of SARS-CoV-2

## TL;DR

This study characterizes a new antibody that targets a conserved region of the SARS-CoV-2 spike protein, offering insights for developing broad coronavirus vaccines.

## Contribution

The study introduces a novel murine monoclonal antibody targeting the conserved stem helix of SARS-CoV-2's S2 domain and compares its mechanism to a known human antibody.

## Key findings

- MAb 7B2 binds to the conserved stem helix of SARS-CoV-2's S2 domain but shows lower neutralization activity compared to S2P6.
- Residues K1149 and D1153 in the S protein form key interactions with MAb 7B2, unlike S2P6.
- The findings suggest conserved epitopes in the S2 domain could be important for pan-CoV vaccine development.

## Abstract

Background/Objectives: For viral entry into host cells, the spike (S) protein of coronavirus (CoV) uses its S1 domain to bind to the host receptor and S2 domain to mediate the fusion between virion and cellular membranes. The S1 domain acquired multiple mutations as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved to give rise to Variant of Concerns (VOCs) but the S2 domain has limited changes. In particular, the stem helix in S2 did not change significantly and it is fairly well-conserved across multiple beta-CoVs. In this study, we generated a murine mAb 7B2 binding to the stem helix of SARS-CoV-2. Methods: MAb 7B2 was isolated from immunized mouse and its neutralization activity was evaluated using microneutralization, plaque reduction and cell–cell fusion assays. Bio-layer interferometry was used to measure binding affinity and AlphaFold3 was used to model the antibody–antigen interface. Results: MAb 7B2 has lower virus neutralizing and membrane block activities when compared to a previously reported stem helix-binding human mAb S2P6. Alanine scanning and AlphaFold3 modeling reveals that residues K1149 and D1153 in S form a network of polar interactions with the heavy chain of 7B2. Conversely, S2P6 binding to S is not affected by alanine substitution at K1149 and D1153 as indicated by the high ipTM scores in the predicted S2P6-stem helix structure. Conclusions: Our detailed characterization of the mechanism of inhibition of 7B2 reveals its distinctive binding model from S2P6 and yields insights on multiple neutralizing and highly conserved epitopes in the S2 domain which could be key components for pan-CoV vaccine development.